High Glucose Regulates The Expression Of Human Na+/dicarboxylate Cotransporter 3 In Renal Proximal Tubularepithelial Cells: Involvement Of PKC, P44/42MAPK

Renal glomerular disease normally combine with tubular interstitial injury,tubular interstitial fibrosis was the common path of various renal diseases developing to the final phrase.It is generally accepted that renal tubular epithelial cells may convert to fibrous cells,and then its energy metabolism disfunction may the vital reason of tubular lesion.Normally,renal proximal tubular epithelial cell is polarized cell,this polarized character is very important to the renal tubular epithelial cell's selective absorption and secretion.Many factors like high glomerular,coagulopathy,ischemie hypoxia,energy metabolism disorder may cause renal proximal tubular epithelial cell's injury,energy metabolism disfunction,then cause insufficient ATP producing,therefore may induce cytoskeleton dissociation,losing the polarized character,finally result in that the renal tubular epithelial cell lose the selective absorption and secretion function.Discarboxylic transporter is an organic anion transport protein family which conveys and reabsorbs the tricarboxylic cycle intermediate products.Sodium-dependent dicarboxylate transporters(NaDC) include low-affinity NaDC1 and high-affinity NaDC3. Despite high similarities structurally and functionally,both are localized to opposite surfaces of renal tubular cells.The molecular mechanisms and localization signals leading to this polarized distribution remain unknown.Na/ discarboxylic transport protein 3 (Na/DC3) is responsible of conveying the tricarboxylic cycle intermediate products from the extracellular fluid,blood to inside of cells.As the Na/DC3 plays an important role in the renal tubular energy metabolism,we presume that various factors,according to effect its expression and construction or polarized distribution,may reduce the energy metabolism disfunction on renal tubular epithelial cells,therefore may play vital role in the renal tubular interstitial lesion.Far from being bystangders in diabetic nephropathy,changes in the proximal tubule are important for the development of progressive diabetic kidney disease.The proximal tubule is uniquely susceptible to a variety of metabolic and emodynamic factors associated with diabetes.Renal function and prognosis correlate better with structural lesions in the tubuli and cortical interstitium than with classical glomerular changes of diabetic nephropathy.On a molecular level,hyperglcaemia and proteins altered by high blood glucose such as Amadori products and Advanced Glycation End-products(AGEs) are key players in the development of diabetic nephropathy,Protein Kinase C(PKC) is the key to open the intracellular signal transport path;Mitogen-Actived Protein Kinase(MAPK) signal path can be activated by:high glucose,sorbitol reaction reduced by high glucose,PKC activation, and Advanced Glycation End-products(AGEs) etc.we investigated the change of NaDC3 in HKC induced by high glucose and its signaling pathways in human kidney tubular cell lines,the effect of high glucose on tubular injure and fibrosis.Method1.Purifying of the polyclonal antibody of hNaDC3 with protein A(Agarose for IgG purification,immobilized GST),evaluation the specificity of antibody by western bloting and immunohistochemistry。2.Using the glucose in different time and concentration to stimulate the HKC cells, comparing with mannitol osmotic pressure,using the North and western bloting to detect the expression changing of hNaDC3's mRNA and protein in a time- and dose-dependent manner after stimulation of high glucose.Fluorscent JC-1 in confocal fluorescence microscopy was used to detect the changes of mitochondrial membrane potential(△Ψ) in norml HKC,25mM high glucose stimulated HKC.Measuring the ATP concentration,H202 production,understand the difference of energy metabolism of the cell in human proximal tubular epithelial cell(HKC).3.Using the glucose in different concentration to stimulate the HKC cells,measuring the expression changing of erk(p44/42 MAPK) To determine whether high glucose stimulates NaDC3 through MAPK and PKC signaling pathways,Pretreated with chelerythrine(a protein kinase A inhibitor),PD98059(a p44/42 mitogen-actived protein kinase(MAPK) inhibitor),SB203580(a p38 MAPK inhibitor),detected expression of hNaDC3 by western bloting.4.Coltured HKC by high glucose long-term,examinated hNaDC3,a- SMA expression variety with western bloting,the cell immunity fluorescence observated a- SMA, Vimentin expression.The full-length hNaDC3(2000bp) was placed into the GFP vector. They were transfected into polarized renal tubule cells lines LLC-PK1 by Lipofectamine reagent(GIBCO).The distribution of hNaDC3 was examined by laser scanning confocal microscopy after these cells were stimulated.Results1.The purified antibody specifically combine with nNaDC3 protein,is located in the bottom of renal proximal tubular.The North and Western bloting results indicate: Glucose upregulated the hNaDC3 expression in a time- and dose-dependent manner and reach the peak at a concentration of 25mM after 48h.Meanwhile ATP concentration distinctly increases as H₂O₂ production increases.HKC stimulated by 25mM high glucose had a high△Ψ,JC-1 monomer was formed in the inner membrane of mitochondria and emitted red fluorescence.The mitochondria membrane electrical potential perceptibly higher than the control group.2.High glucose stimulation will increase the HKC cell p-erk expression,erk activity and hNaDC3 high expression is obviously related;erk idiocratic inhibitor PD98059, Chelerythrine can restrain the high hNaDC3 expression effected by glucose,but p38 idiocratic inhibitor PD203580 has no effect.3.Using laser scanning confocal fluorescence microscope to observe LLC-PK1 cells which were transfected with pGFP-hNaDC3 and stimulated by high glucose,24 hr pGFP-hNaDC3 fluorescent distribution indicates a no-polarity change,Both the ceiling membrane and bottom membrane have expression;48hr fluorescence obviously fade.To the HKC with the long-term cultivation,Western measured hNaDC3 expression is obviously lower than normal group,but a-SMA expression increased.Meanwhile,cell immunity fluorescence result showed HKC expression Vimentin increasing,appearance of a-SMA expression.ConclusionThe hNaDC3 high expression,which was induced by high glucose stimulation on HKC in short-term,shows a time- and dose-dependent manner.At the same time,high glucose stimulation obviously enhanced the cell energy metabolism,and tranduced through erk activity and PKC signal path.Long-term stimulation decreased the HKC hNAdc3 expression,hNaDC3 no-polarity distribution with appearance of Vimentin and a-SMA expression,confirmed the appearance of HKC cells transdifferentiation.As a result of this research,the high glucose caused the hNaDC3 expression increasing in short-term, meantime,the increasing of ATP concentration,H₂O₂ producing,and mitochondria membrane electrical potential farther confirmed that hNaDC3 took part in the energy metabolism,may play an important role on renal tubular epithelial cells transdifferentiation which caused by high glucose,and then after the transdifferentiation,that cell polarity disappeared and membrane protein desquamated may be the reason which induced the decreaseing of hNaDC3 expression.